Characterization of the tumor suppressor Usp9x in pancreatic ductal organoids and pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death due to cancer in the United States. The five-year survival rate for PDAC patients is the lowest among common cancers, and remains at only 6% despite intensive efforts to improve clinical prognosis. The conventional tissue culture methods to grow pancreatic cancer cell lines in monolayer allow us to dissect molecular pathways and drug responses in PDAC. However there is a significant translational gap between in vitro and in vivo. For example, when orthotopically injected, PDAC cell lines grown in monolayer do not produce a stromal reaction, unlike tumors from genetically engineered mouse models (GEMMs) or human patients. Moreover it is a daunting task to utilize GEMMs to validate the function of genes and the responses of drugs in a high throughput manner. Therefore, it is urgently required to bridge the gap between in vitro and in vivo models. Here, I propose to establish three dimensional culture and transplantation models to study PDAC progression and characterize gene function in primary pancreatic ductal cells. I have successfully grown murine normal, premalignant and malignant pancreatic ductal epithelial cells in three dimensional semisolid matrices (called pancreatic organoids hereafter) from GEMMs (wild-type, Kras+/LSL-G12D, Pdx1-Cre; Kras+/LSL-G12D or Pdx1-Cre;Kras+/LSL-G12D; Trp53+/LSL-R172H). More importantly, upon orthotopic transplantation, engrafted organoid cells faithfully recapitulate PDAC progression unlike 2D cell lines. Thus, murine orthotopically grafted organoid (OGO) models will provide a unique platform to study PDAC progression, biomarker discovery and functional validation of therapeutic targets. Previously, we have identified the deubiquitinating enzyme, Usp9x as a new tumor suppressor in PDAC using Sleeping Beauty insertional mutagenesis screening. We found that cells lacking Usp9x had an increased resistance to apoptosis following matrix detachment, and we implicated the E3 ubiquitin ligase Itch, a substrate of Usp9x in this process. However, the role of Usp9x and Itch in PDAC remains largely unknown. Using three dimensional pancreatic ductal organoids, I will study the role of Usp9x and its substrate Itch in PDAC progression, allowing us to investigate the role of Usp9x and Itch in normal and premalignant pancreatic ductal cells. Usp9x and Itch have protein targets that can explain their tumor suppressor properties. These target proteins will be identified by whole proteome and Ubiquitin-proteome methods. To characterize the functions of Usp9x and Itch, the role of individual substrates of Usp9x and Itch will be investigated using OGO mice. The newly identified substrates of Usp9x and Itch will elucidate new tumor suppressive pathways in PDAC progression, paving the road towards new therapeutics.